Detection of histone modification in cell-free nucleosomes

ABSTRACT

This invention relates to the diagnosis of disease conditions, such as cancer and autoimmune disease, by the analysis of cell-free nucleosomes in samples from individuals. Methods of the invention may include contacting cell-free nucleosomes from a biological fluid sample obtained from the individual with an antibody that binds specifically with a modified histone protein. Binding of the antibody to the nucleosomes is indicative that the individual has the disease condition.

This application is a continuation application of application Ser. No.10/568,691 (U.S. Patent Application Publication No. 2007-0160989 A1),filed Aug. 30, 2006 (allowed), which is a U.S. national phase ofinternational application PCT/GB2004/003564, filed 18 Aug. 2004, whichdesignated the U.S. and claims priority of GB 0319376.0, filed 18 Aug.2003, the entire contents of each of which are hereby incorporated byreference.

This invention relates to the diagnosis of disease conditions, such ascancer and autoimmune disease, by the analysis of cell-free nucleosomesin samples from individuals, in particular the analysis of cell-freenucleosomes containing histone modifications.

In eukaryotes, DNA is complexed with proteins to form nucleosomes, thebasic sub-unit of chromatin. Nucleosomes consist of approximately 150DNA bade pairs wrapped around a histone core, which is a protein complexinvolving the four histones H4, H3, H2B and H2A. The amino-terminaltails of these proteins are among the most evolutionary conservedproteins known. These tails are post-translationally modified by theaddition of a range of chemical groups including methyl, acetyl andphosphoryl. These chemical modifications, or marks, play a key role indetermining chromatin structure and hence access to the cells genomicDNA (Wu J and Grunstein M (2000) Trends Biochem. Sci. 25, 619-623;Berger SL (2001) Oncogene 20, 3007-3013). It has also been shown thatthe marks are involved in the control mechanism for a wide range ofcellular processes. For example, in general, deacetylation of marks andcertain methylation marks are associated with gene silencing (Hu J F andHoffman A R (2001) Methods Mol Biol 181, 285-296; Rice J C and Allis C D(2001) Curr Opin Cell Biol 13, 263-273; Carrozza M J et al (2003) TrendsGenet 19, 321-329; Nephew K P and Huang T H (2003) Cancer Lett 190,125-133) and phosphoryl marks with apoptosis (Enomoto R et al (2001) MolCell Biol Res Commun 4, 276-281; Ajiro K (2000) J Biol Chem 275,439-443; Talasz H, et al (2002) Cell Death Differ 9, 27-39; Rogakou EPet al (2000) J Biol Chem 275, 9390-9395) and mitosis (Crosio et al(2002) Mol Cell Biol 22 874-885; Goto et al (2002) Genes Cells 7, 11-17;Hans and Dimitrov (2001) Oncogene 20, 3021-3027; Preuss et al (2003)Nucl Acids Res 31, 878-885). Nucleosomes marked in a specific manner canbe isolated from cells by using specific antibodies, and the DNAcomponent analysed (for example, Clayton et al (2000) EMBO J 19,3714-3726; Li et al (2001) Mol Cell Biol 21, 8213-8224; Osano and Ono(2003) Eur J Biochem 270, 2532-2539; Kondo and Issa (2003) J Biol Chem(2003) 278(30): 27658-62).

Patients suffering from conditions, such as cancer and autoimmunedisease, have nucleosomes circulating in the blood resulting fromincreased apoptosis (Holdenrieder et al (2001) Int J Cancer 95, 114-120;Trejo-Becerril et al (2003) Int J Cancer 104, 663-668; Kuroi et al 1999Breast Cancer 6, 361-364; Kuroi et al (2001) Int J Oncology 19, 143-148;Amoura et al (1997) Arth Rheum 40, 2217-2225; Williams et al (2001) JRheumatol 28, 81-94). Measurement of the levels of cell-free nucleosomeshas been proposed as a means of diagnosing diseases associated withapoptosis (Holdenrieder et al (1999) Anticancer Res. 19, 2721-2724).However, the presence of cell-free nucleosomes with specific marks wasnot assessed.

The present invention relates to methods for detecting nucleosomescontaining modified histones in samples from patients, in particularmethods that involve antibody-antigen interactions.

Various aspects of the invention relate to the use antibodies whichspecifically bind to modified histones to detect nucleosomes in sampleswhich comprise modified histones.

One aspect of the invention provides a method of assessing a diseasecondition in an individual comprising;

-   -   contacting said nucleosomes from a biological fluid sample        obtained from the individual with an antibody which binds        specifically with a modified histone protein,    -   wherein binding of said antibody to said nucleosomes is        indicative that the individual has a disease condition.

A disease condition in the individual may be assessed by determining oneor more of: the presence of one or more histone modifications in thesample, an increase in the number of cell-free nucleosomes containingmodified histones in the sample relative to normal levels, an alterationin the ratio of one or more particular histone modifications relative toanother histone modifications in the sample and a threshold number ofnucleosomes in the sample which comprise a histone modification.

After the biological fluid sample has been contacted with the antibodyunder conditions suitable to allow specific binding of the antibody toits target antigen, nucleosomes comprising the modified histone may beidentified and, optionally, isolated using standard techniques.

A biological fluid suitable for use in accordance with the presentmethods may include sera, plasma, lymph, blood, blood fractions, urine,synovial fluid, spinal fluid, saliva, and mucous. Blood, serum or plasmaare preferred.

Nucleosomes may be concentrated from the biological fluid sample beforecontact with the antibody. Nucleosomes may be concentrated from thesample of biological fluid by any convenient concentration method,including, for example:

-   -   centrifugal filtration such as centrifugal filtration units with        an appropriate molecular weight cut-off membrane e.g.        Millipore's Centricon® or Amicon® units,    -   acid precipitation (Yoshida, M et al, (1990), J Biol Chem 265,        17174-17179).    -   immunoprecipitation using conventional methods, for example, by        incubating the sample with an anti-nucleosome antibody or a        histone mark-specific antibody, and then immunopurifying the        antibody/antigen complex using a spin column packed with an        immunoaffinity matrix. The captured nucleosomes would then be        eluted and analysed.    -   Separation based on charge, for example, binding to polyK coated        solid supports (Williams R C et al,(2001) J Rheumatol 28,        81-94).    -   Separation based on biotinylation. Histones can be biotinylated        by biotinidase (Hymes J et al (1995), Biochem Mol Med 56, 76-83;        Stanley J S et al, (2001) Eur J Biochem 268, 5424-5429.

In some embodiments, nucleosomes may be concentrated by a method otherthan collection on a poly K or streptavidin-coated support.

A histone mark may be a post-translational chemical change to one ormore histone amino acid residues, for example addition/removal of achemical group or isomerisation of an amino acid residue.

An antibody specific for a modified histone is specific for a uniqueepitope formed by post-translational modification of a core histone, forexample histone H2A, H2B, H3, H4 (Luger, K. et al (1997) Nature 389,251-260) or a modification or variant thereof (see for example (Ausio J(2001) Biochem Cell Bio 79, 693). Known sequences of histones aredescribed in the NHGRI/NCBI histone sequence database which isaccessible on-line.

A modification may be in the central region of a histone or in theflexible N-terminal or C-terminal tail.

Post-translational modification may include acetylation, methylation,which may be mono-, di- or tri-methylation, phosphorylation,ribosylation, citrullination, ubiquitination, hydroxylation,glycosylation, nitrosylation, glutamination and/or isomerisation (AusioJ (2001) Biochem Cell Bio 79, 693).

A lysine residue which is methylated may be mono-, di- ortri-methylated. An arginine residue which is methylated may besymmetrically or asymmetrically dimethylated, or monomethylated.

An histone amino acid residue having a modification may be any Ser, Lys,Arg, His, Glu, Pro or Thr residue within the histone amino acidsequence.

For example, a lysine residue within the core histone sequence may bemono-, di- or tri-methylated, acetylated or ubiquitinated, an arginineresidue within the core histone sequence may be monomethylated,symmetrically or asymmetrically dimethylated or converted to citrulline,a serine or threonine residue within the core histone sequence may bephosphorylated and/or a proline residue within the core sequence may beisomerised.

The notation used to describe a particular histone modificationindicates which histone has been modified, the particular amino acid(s)that have been modified and the type of modification that has occurred.For example H3 Lys 9(Me) denotes the methylation of histone H3 at lysine9.

Examples of modifications include modifications shown in table 1.

A histone mark which produces a cellular effect may consist of onemodification to a histone or may consist of two or more histonemodifications. In other words, a single mark, which may for example beassociated with silencing or activation, may consist of a combination ofseparate modifications to different residues within a histone sequence.

For example, a modified histone may comprise a mark which is associatedwith gene silencing, such as H3 Lys 9(Me) H3 Lys 27(Me), H3 Lys 36(Me),H3 Lys 79(Me) and H4 Lys 20(Me) or a mark which is associated with geneactivation, such as H3 Lys 4(Me) H3 Lys 9(Ac), H3 Lys 14(Ac) and H3 Lys23(Ac)

Antibodies which are specific for histone marks that are associated withactive gene sequences (euchromatin) or inactive gene sequences(heterochromatin) may be used, for example, to detect inappropriate geneexpression which is indicative of a disease state. Screening thepopulation of cell-free nucleosomes present in a sample from anindividual may reveal the inactivation of a tumour suppression gene oralternatively, the activation of an oncogene.

A ‘modified nucleosome’ is a nucleosome which comprises a histonecomprising one or more modifications as described above.

An antibody which specifically binds to an antigen such as a modifiedhistone or nucleosome may not show any significant binding to moleculesother than the antigen. An antibody may specifically bind to aparticular epitope which is carried by a number of antigens, in whichcase the antibody will be able to bind to the various antigens carryingthe epitope.

In some embodiments, a disease condition may be assessed by determiningthe presence of two or more histone modifications in cell-freenucleosomes in the sample. In particular, the presence of a histone markconsisting of more than one modification may be determined bydetermining the presence of the two or more separate modifications. Twoor more histone modifications in a sample may be characterised bycontacting the sample with an antibody that specifically binds to two ormore histone modifications or alternatively, contacting the sample withtwo or more antibodies, each antibody specifically binding to adifferent histone modification.

Another aspect of the invention comprises a method of assessing histonemodification in cell-free nucleosomes in a biological fluid samplecomprising;

-   -   contacting a biological fluid sample with an antibody which        binds specifically to a histone comprising a modification; and,    -   determining the binding of said antibody to nucleosomes in said        sample,    -   the binding of said antibody being indicative of the presence of        modified histone in nucleosomes in the blood of said individual.

An antibody may specifically bind to a histone modification describedabove, for example a modification shown in Table 1, or a combination ofsuch modifications.

In some preferred embodiments, an antibody may bind specifically to ahistone comprising a modification shown in Table 2 or a combination ofsuch modifications.

In some embodiments, the biological fluid sample may be contacted with afurther antibody which binds specifically to histone comprising adifferent modification from the first antibody. A range of antibodiesmay be employed to detect the presence of a range of histonemodifications.

Cell-free nucleosomes in fluid samples from patients may be used toassess disease conditions associated with cell death, in particularcancer and/or autoimmune disease. For example, the presence of cancercells in an individual may generate a higher level of cell freenucleosomes in the blood as a result of the increased apoptosis of thecancer cells. An antibody directed against marks associated withapoptosis, such as H2B Ser 14(P), may be used to selectively isolatenucleosomes that have been released from apoptotic neoplastic cells.

Another aspect of the invention provides a method of assessing a diseasecondition in an individual comprising;

-   -   contacting biological fluid sample obtained from an individual        with an antibody which binds specifically to a modified histone,    -   determining the binding of said antibody to nucleosomes in said        sample,    -   the binding of said antibody to nucleosomes in said sample being        indicative that said individual has a disease condition.

A modified histone may, for example, have a modification selected fromthe group consisting of H2B Ser 14 (Phos), H3 lys 9(Me), H3 lys 27(Me)and H3 Ser 10 (Phos). In some embodiments, the modified histone is notH2B Ser 14(Phos).

Diseases associated with modified, cell-free nucleosomes include, butare not limited to, pre-malignant and malignant neoplasms and tumours,(e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g., lungcancer, small cell lung cancer, gastrointestinal cancer, bowel cancer,colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer,testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreascancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma,melanoma), leukemias, autoimmune diseases (e.g. systemic lupuserythematosus) and proliferative disorders (e.g. psoriasis, bonediseases, fibroproliferative disorders of connective tissue, cataractsand atherosclerosis).

A pre-malignant or malignant condition may occur in any cell-type,including but not limited to, lung, colon, breast, ovarian, prostate,liver, pancreas, brain, and skin.

An antibody that specifically binds to a modified histone may begenerated using techniques which are conventional in the art. Methods ofproducing antibodies include immunising a mammal (e.g. mouse, rat,rabbit, horse, goat, sheep or monkey) with a modified histone or apeptide fragment of the histone which comprises the modification ormark. Peptide fragments with particular modifications can be designedfrom known histone sequences and produced by routine synthesis methods.Antibodies may be obtained from immunised animals using any of a varietyof techniques known in the art, and screened, preferably using bindingof antibody to antigen of interest. For instance, Western blottingtechniques or immunoprecipitation may be used (Armitage et al., (1992)Nature 357, 80-82).

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith any of the proteins (or fragments), or may be one constructed usingsequences obtained from an organism which has been exposed to theantigen of interest.

Antibodies suitable for use in accordance with the present methods arealso available from commercial suppliers.

The binding of an antibody may be determined by any appropriate means.Tagging with individual reporter molecules is one possibility. Thereporter molecules may directly or indirectly generate detectable, andpreferably measurable, signals. The linkage of reporter molecules may bedirectly or indirectly, covalently, e.g. via a peptide bond ornon-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

Radioimmunoassay (RIA) is another possibility. Radioactive labelledantigen is mixed with unlabelled antigen (the test sample) and allowedto bind to the antibody. Bound antigen is physically separated fromunbound antigen and the amount of radioactive antigen bound to theantibody determined. The more antigen there is in the test sample, theless radioactive antigen will bind to the antibody. A competitivebinding assay may also be used with non-radioactive antigen, usingantigen or an analogue linked to a reporter molecule. The reportermolecule may be a fluorochrome, phosphor or laser dye with spectrallyisolated absorption or emission characteristics. Suitable fluorochromesinclude fluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colour or causechanges in electrical properties, for example. They may be excitable,such that electronic transitions between energy states result incharacteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed. The mode of determining binding is not a feature of thepresent invention and those skilled in the art are able to choose asuitable method according to their preference and general knowledge.

The signals generated by individual antibody-reporter conjugates may beused to derive quantifiable absolute or relative data of the relevantantibody binding in samples (normal and test).

Methods of the invention may be carried out in any convenient format.Immunological assays are well-known in the art and many suitable formatsare available, for example ELISA, Western blotting, or Biacore®,(Biacore, Upsala, Sweden). In some preferred embodiments, a sandwichassay format may be employed.

A sandwich assay employs a capture antibody and a detection antibody todetect the presence of antigen in a sample. The capture antibody may,for example, bind specifically to a nucleosome and the detectionantibody to a histone with a particular modification, or vice versa.

Another aspect of the invention provides a method of assessing histonemodification in cell-free nucleosomes in a biological fluid sample froman individual comprising;

-   -   contacting a biological fluid sample from said individual with a        first antibody; and,    -   determining binding of said first antibody to a nucleosome        comprising a histone modification using a second antibody,    -   wherein one of said first or second antibodies binds to a        nucleosome and the other of said first or second antibodies        binds specifically to a modified histone.

In some embodiments, the first antibody binds to nucleosomes and thesecond antibody binds specifically to the modified histone. A method ofassessing histone modification in nucleosomes in a biological fluidsample from an individual may thus comprise;

-   -   contacting a biological fluid sample from said individual with a        first antibody which binds to nucleosomes; and,    -   determining the presence of a modified histone in a nucleosome        bound by said first antibody using a second antibody which binds        specifically to a modified histone.

Antibodies which bind specifically to modified histones are described inmore detail above. An antibody which binds to a nucleosome may bind toany epitope commonly found on any unmodified component of thenucleosomes, including histone and non-sequence specific DNA epitopes.In some embodiments, an antibody may bind to both the histone and DNAcomponents of the nucleosome. An antibody may bind specifically to oneor more nucleosome components.

Suitable anti-nucleosome antibodies include the antibody known as clone11E6 (available from B D PharMingen) which interacts with the(H2A-H2B)-DNA sub-nucleosomal complex (Jovelin F et al (1998) Eur JImmunol 28, 3411).

In other embodiments, the second antibody binds to nucleosomes and thefirst antibody binds specifically to the modified histone. A method ofassessing histone modification in nucleosomes in a biological fluidsample from an individual may thus comprise;

-   -   contacting a biological fluid sample from said individual with a        first antibody which binds specifically to a modified histone,    -   determining the binding of said first antibody to a nucleosome        comprising a modified histone using a second antibody which        binds to a nucleosome.

One of said first and second antibodies may be immobilised and thebinding of the other antibody may be detected. Preferably, the firstantibody is immobilised. An antibody may be immobilised, for example, byattachment to an insoluble support. The support may be in particulate orsolid form and may include a plate, a test tube, beads, a ball, a filteror a membrane. An antibody may, for example, be fixed to an insolublesupport that is suitable for use in affinity chromatography. Methods forfixing antibodies to insoluble supports are known to those skilled inthe art. An antibody may be immobilised, for example, to isolatecell-free nucleosomes from the biological fluid sample.

The non-immobilised antibody may comprise a detectable label asdescribed above. For example, the antibody may be labeled with afluorophore such as FITC or rhodamine, a radioisotope, or a non-isotopiclabeling reagent such as biotin or digoxigenin; antibodies containingbiotin may be detected using “detection reagents” such as avidinconjugated to any desirable label such as a fluorochrome.

In some embodiments, the non-immobilised antibody may be detected usinga third antibody which binds to said non-immobilised antibody. Asuitable third antibody is labelled and is binds specifically to thefirst or second antibody. The third antibody may comprise a detectablelabel.

In some embodiments, a blocking reagent may be used to block or absorbinterfering endogenous components, such as antibodies or proteins. Forexample, samples may be depleted of endogenous antibodies by, forexample, application to a spin column packed with an immunoaffinitymatrix to remove immunoglobulin. Alternatively, the potentialinterference by heterophilic antibodies could be minimised by the use ofa blocking reagents. Suitable blocking reagents are availablecommercially, for example, HBR from Scantibodies Ltd (Santee, Calif.,US)). Excess albumin in samples may conveniently be depleted by using analbumin affinity spin column (Montage™ Albumin Deplete kit, Millipore).

Antibodies specific for modified histones may be used to detect anyabnormal modifications that would indicate a disease state.Alternatively, the nucleic acid sequences associated with modifiednucleosomes may be analyzed using standard techniques to assess adisease condition or susceptibility to a disease condition.

Methods as described herein may be used to isolate and/or identitynucleic acid sequences associated with a particular mark. These nucleicacid sequences may be associated with a disease condition. Identifyingthe DNA associated with modified nucleosomes may also be useful inmonitoring the progress of a therapeutic treatment, for example,monitoring positive and/or adverse effects resulting from treatment.

Methods of the invention may comprise isolating a nucleosome comprisinga modified histone. Nucleosomes comprising modified histones may beisolated by immunoprecipitation using a modified histone-specificantibody or a nucleosome specific antibody as described herein.Alternatively, nucleosomes may be isolated by binding to an immobilisedantibody, as described above.

Once the nucleosomes have been isolated from the sample, the DNAassociated with the nucleosomes can be recovered using standardtechniques. For example, DNA may be immobilised onto filters, columnmatrices, or magnetic beads. Numerous commercial kits, such as theQiagen QIAamp kit (Quiagen, Crawley, UK) may be used. Briefly, thesample may be placed in a microcentrifuge tube and combined withProteinase K, mixed, and allowed to incubate to lyse the cells. Ethanolis then added and the lysate is transferred to a QIAamp spin column fromwhich DNA is eluted after several washings. Optionally, the isolated DNAmay be amplified through PCR or other amplification techniques. Thesequence of the nucleosome-associated DNA may be obtained, for exampleto identify the polypeptide encoded by the DNA. Nucleosome associatedDNA may be associated with a particular histone mark or modification.For example, depending on the binding specificity of the antibody usedto initially isolate the nucleosomes from the sample, genes may beidentified that are associated with activation or silencing marks.

Any of analytical procedures known to those skilled in the art may beused to identify the DNA sequences associated with isolated nucleosomes.DNA sequences may, for example, be identified by direct microsequencingof the purified DNA.

Alternatively, the purified DNA may be first amplified using PCRtechnology or other amplifying technique before further analysis of theDNA.

In some embodiments, the DNA associated with the isolated nucleosomesmay be identified by contacting the purified DNA with known nucleic acidsequences under conditions suitable for hybridisation of complementarysequences, wherein hybridisation of the purified DNA to its complementidentifies the purified DNA sequence; and determining hybridisation. Forexample, Southern Blot analysis may be conducted in which either theknown DNA sequences or the purified DNA serves as the labelled probe,and the unlabeled sequences are immobilized on a solid surface.Formation of nucleic acid duplexes is then detected. Thenucleosome-associated DNA can then be identified from the sequence(s) towhich it hybridises.

Nucleic acid probes can be labelled with a detectable marker usingstandard techniques known to those skilled in the art. For example thenucleic acid probes can be labelled with a fluorophore, a radioisotope,or a non-isotopic labelling reagent such as biotin or digoxigenin.

Known nucleic acid sequences, for example, sequences from various genesof interest, may be immobilized on a solid surface, as described above.Preferably, the sequences are immobilized in the form of a microarray,in which each known sequence is assigned a position on a solid surface.Preferably, the microarray comprises a plurality of DNA molecules, eachhaving a different known sequence. The purified nucleosome DNA may belabelled and then placed in contact with a microarray of known sequencesunder conditions suitable for the hybridisation of complementarysequences. After a predetermined length of time the unbound andnon-specifically bound material may be washed from the microarray andthe array I screened for detectable signals. A signal generated at aspecific position on the solid surface by hybridisation of a purifiednucleosome DNA sequence to its complement, identifies the purifiednucleosome DNA sequence.

Microarrays allow miniaturisation of assays, e.g. making use of bindingagents (such as nucleic acid sequences) immobilised in small, discretelocations (microspots) and/or as arrays on solid supports or ondiagnostic chips. These approaches can be particularly valuable as theycan provide great sensitivity (particularly through the use offluorescent labelled reagents), require only very small amounts ofbiological sample from individuals being tested and allow a variety ofseparate assays to be carried out simultaneously. This latter advantagecan be useful as it provides an assay for a number of differentsequences to be carried out using a single sample. Examples oftechniques enabling this miniaturised technology are provided inWO84/01031, WO88/1058, WO89/01157, WO93/8472, WO95/18376/WO95/18377,WO95/24649 and EP-A-0373203, the subject matter of which are hereinincorporated by reference.

The principles of microarray hybridisation are described in Yershov, G.et al (1996) Proc Natl Acad Sci USA 93 4913-4918, Cheung V. G. et al(1999) Nature Genetics 21 15-19, and Schena, M. (1999) DNA Microarrays“a practical approach”, ISBN, 0-19-963777-6, Oxford press, editor B. D.Hames. In brief, the DNA microarray may be generated usingoligonucleotides that have been selected to hybridise with the specifictarget polymorphism. These oligonucleotides may be applied by a robotonto a predetermined location of a glass slide, e.g. at predetermined X,Y cartesian coordinates, and immobilised. The sample RNA or DNA (e.g.fluorescently labelled RNA or DNA) is introduced on to the DNAmicroarray and a hybridisation reaction conducted so that sample RNA orDNA binds to complementary sequences of oligonucleotides in asequence-specific manner, and allow unbound material to be washed away.Sequences can thus be identified by their ability to bind tocomplementary oligonucleotides on the array and produce a signal. Theabsence of a fluorescent signal for a specific oligonucleotide probeindicates that the sequence of the sample DNA or RNA is not present onthe micoarray. Of course, the method is not limited to the use offluorescence labelling but may use other suitable labels known in theart. Fluorescence at each coordinate can be read using a suitableautomated detector, in order to correlate each fluorescence signal witha particular oligonucleotide.

Hybridisation of nucleosome associated DNA from said individual may becompared with the hybridisation of nucleosome associated DNA from otherindividuals. For example, hybridisation patterns from a patient with aproliferative disorder may be compared with patterns from a healthyindividual to identify genes whose chromatin is differentially marked(for example, activated or inactivated) in the proliferative disorder.For example, a tumour suppressor gene may be associated with a silencingmark or an oncogene with an activation mark in a cancer condition.

An aspect of the invention provides a method of identifying a tumoursuppressor gene comprising;

-   -   contacting biological fluid sample obtained from an individual        having a cancer condition with an antibody which binds        specifically to a histone having a modification associated with        silencing,    -   isolating nucleosomes bound to said antibody, sequencing DNA        associated with said bound nucleosomes; and,    -   identifying said DNA as a tumor suppressor gene.

A method may comprise comparing said DNA with DNA associated with saidbound nucleosomes in sample from a healthy individual (i.e. anindividual not having a cancer condition). A DNA sequence which isassociated with a silencing mark in the cancer sample but not thenon-cancer sample is a candidate tumour suppressor.

In some embodiments, a modification associated with silencing mayexclude Lys 9 methylation of histone H3.

A method may include concentrating the nucleosomes in the sample by amethod other than collection on a poly K or streptavidin-coated support,prior to contacting with the antibody.

A method of identifying a tumour suppressor gene may include contactingthe nucleosomes with a first antibody which binds specifically to ahistone having a modification associated with silencing and a secondantibody which binds to nucleosomes, for example in a sandwich assayformat.

An aspect of the invention provides a method of identifying an oncogenecomprising;

-   -   contacting biological fluid sample obtained from an individual        suffering from a cancer condition with an antibody which binds        specifically to a histone having a modification associated with        activation,    -   isolating nucleosomes bound to said antibody,    -   sequencing DNA associated with said bound nucleosomes, and;    -   identifying said DNA as an oncogene.

Modifications associated with gene activation are described in moredetail above. In some embodiments, a modification associated withactivation may exclude H3 Lys 4 (Me), H3 Lys 9 (Ac) and/or H4 Lys 5(Ac).

A method may comprise comparing said DNA with DNA associated with saidbound nucleosomes in sample from a healthy individual (i.e. anindividual not having a cancer condition). A DNA sequence which isassociated with an activation mark in the cancer sample but not thenon-cancer sample is a candidate oncogene.

A method may include concentrating the nucleosomes in the sample by amethod other than collection on a polyK or strepavidin-coated support,prior to contacting with the antibody.

A method of identifying an oncogene may include contacting thenucleosomes with a first antibody which binds specifically to a histonehaving a modification associated with silencing and a second antibodywhich binds to nucleosomes, for example in a sandwich assay format.

Methods described herein may be useful in detecting chromatinalterations which are associated with a disease condition. Cell-freenucleosomes may be isolated from samples from healthy individuals andfrom individuals having a disease condition, using a modified histonespecific antibody and optionally a nucleosome specific antibody, togenerate a first and second pool of nucleosomes, respectively.Preferably, methods of detecting chromatin alterations associated withdisease comprise contacting the nucleosomes with a first antibody whichbinds specifically to a histone having a modification and a secondantibody which specifically binds to nucleosomes, for example in asandwich assay format.

After isolation, the nucleic acid associated with the isolatednucleosomes may be isolated and/or purified from the first and secondpools of nucleosomes to generate a first and second pool of purifiednucleic acid. The purified nucleic acid in each pool is then analyzed,using standard molecular techniques such as DNA sequencing, nucleic acidhybridization analysis (including Southern blot analysis), PCRamplification or differential screening, to identify differences betweenthe two pools of nucleic acid sequences. Those nucleic acid sequencesthat are present in only one of the two pools represent nucleic acidsequences that are potentially related to the disease condition.

For example, the pools of nucleic acid sequences may be separatelycontacted with identical sets of DNA microarrays under conditions thatallow for hybridization between complementary sequences. The microarraysmay, for example, contain a subset of sequences that are associated withparticular diseases (such as various known oncogene and tumor suppressorgenes) or may contain the entire set of expressed sequences for one ormore particular cell types and developmental stages. Hybridisationbetween a sequence in the pool of nucleosome associated nucleic acid anda nucleic acid sequence immobilised within the microarray produces adetectable signal, which allows the nucleosome associated nucleic acidto be identified. Suitable microarrays can be prepared using techniquesknown to those skilled in the art.

In some embodiments, the pools of nucleosome-associated nucleic acid maybe amplified by PCR and/or labelled prior to contacting them with themicroarray. Washing of the microarray removes non-bound andnon-specifically bound material and allows detection of the labelledsequences that have specifically hybridised to sequences present on themicroarray, thus identifying of the labelled sequences. Comparison ofthe hybridisation patterns obtained with the first and second pools ofnucleosome-associated nucleic acid allows the identification ofchromatin alterations that are potentially associated with a diseasecondition.

Pools of nucleosomes may be compared using a gene chip, DNA microarray,or a proteomics chip using standard techniques known to those skilled inthe art (For example, WO 01/16860,WO 01/16860, WO 01/05935, WO 00/79326,WO 00/73504, WO 00/71746 and WO 00/53811).

Methods as described herein also allow the identification of genomic DNAwhich is associated with particular markers. DNA which is associatedwith a nucleosome having a particular histone modification may, forexample, be immobilized on a solid surface or “chip”. This DNA may, forexample, represent all the nucleic acid sequences of a given cell thatis competent for transcription or not competent for transcription,depending on the histone modification (for example, active: H3 lys 4(Me), inactive: H3 lys 9 (Me).

Other aspects of the invention relate to the identification andmonitoring of patients having disease conditions which are associatedwith the aberrant marking of histones.

A method of identifying a patient as a responsive to histonemodification modulation therapy may comprise;

-   -   determining the level of histone modification in cell-free        nucleosomes within a sample obtained from the patient, relative        to a sample obtained from a healthy individual,    -   a change, for example an increase or decrease, in the level of        modification being indicative that the patient is responsive to        histone modification modulation therapy.

Methods of the invention may also be used to monitor the effect ofhistone modification modulation therapy. Histone modification modulationtherapy may include, for example, inhibition of histone modifying orde-modifying enzymes, such as histone methyl transferases, acetylasesand deacetylases.

A method of monitoring the effect of histone modification modulationtherapy in a patient may comprise;

-   -   contacting samples obtained from the patient at first and second        time points in said therapy with an antibody which specifically        binds to a histone having a modification; and    -   determining binding of said antibody to said samples;    -   a change, for example an increase or decrease, in the binding of        said antibody to the sample obtained at the second time point        relative to the first being indicative of the effect of said        therapy.

A patient may be suffering from a cancer or autoimmune condition, asdescribed above.

For example, tumour cells may over-express enzymes that remove acetylmarks, leading to reduced expression of tumour suppressor genes(Johnstone R W (2002) Nature Reviews Drug Discovery, 1, 287). Patientsidentified using the present methods as having reduced histoneacetylation may be treated with an agent which inhibits histonedeacetylating enzymes. This increases histone acetylation, therebyincreasing expression of tumor suppressor genes. The effect of therapymay be monitored by determining an increase in the level or amount ofacetylation marks.

Aurora kinase B, which phosphorylates of H3 Ser 10, is over-expressed inmany cancer conditions. Aurora kinase B inhibitors have been shown tohave an anti-proliferative effect which is associated with inhibition ofthis histone marking step (Ditchfield C (2003) J. Cell Biol. 161,267).Patients with increased phosphorylation at H3 Ser 10 may be identifiedusing methods of the invention and the effects of treatment with anaurora kinase inhibitor monitored.

Other aspects of the invention relate to the analysis of the DNAassociated with specifically marked nucleosomes in order to identify theappropriate treatment regimes.

For example, such analysis may indicate the propensity of a tumour tometastasise, the hormone dependence of a tumour, or the activation in atumour of certain resistance genes and pathways, for example,glutathione S-transferase-pi(Townsend D and Tew K (2003) Am JPharmacogenomics 3, 157-172), multidrug resistance associated protein,p-glycoprotein (Mattern J (2003) Anticancer Res 23, 1769-1772) andglyoxalase-I(Tsuruo T (2003) Cancer Sci 94, 15-21). The effect oftreatment regimes could be monitored, for example, by observing changesin gene silencing/activation marks associated with these genes.

-   -   A method of assessing a patient for a therapeutic treatment may        comprise;    -   determining the presence of one or more genes which confer        resistance to said treatment in a cell-free nucleosome in a        sample obtained from the patient, as described above,    -   wherein said nucleosome comprises or contains a histone        modification associated with activation or silencing.    -   Histone modifications associated with activation or silencing        are described in more detail above.    -   Various further aspects and embodiments of the present invention        will be apparent to those skilled in the art in view of the        present disclosure. All documents referenced in this        specification are incorporated herein by reference.    -   All combinations and sub-combinations of the features described        above, whether or not specifically described or exemplified, are        encompassed by the invention.    -   Certain aspects and embodiments of the invention will now be        illustrated by way of example and with reference to the figures        and table described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the efficient recovery and detection of marked(dimethylated Lys 4 of histone H3) chicken nucleosomes spiked into humanblood. Column A shows platelet poor plasma (PPP) derived from normalblood spiked with chicken nucleosomes, column B shows buffer spiked withchicken nucleosomes and column C shows PPP derived from normal blood.

FIG. 2 shows an ELISA standard curve for chicken nucleosomes spiked intobuffer, detected using an antibody to dimethylated Lys 4 of histone H3.

FIG. 3 shows the analysis of a concentrated plasma sample from a patientwith cancer and of a concentrated normal plasma sample using an antibodyto dimethylated Lys 4 of histone H3.

Table 1 shows a list of known histone marks. In the table, Me=mono, dior trimethyl, Ac=Acetyl, Phos=Phosphorylation, Ubiq=Ubiquitinated (ForArg, Me can mean mono or dimethylated, where dimethylation can besymmetrical or asymmetrical).

Table 2 shows examples of preferred marks according to the invention. Asfor table 1, Me=mono, di or trimethyl, Ac=Acetyl, Phos=Phosphorylation,Ubiq=Ubiquitinated (For Arg, Me can mean mono or dimethylated, wheredimethylation can be symmetrical or asymmetrical).

Table 3 shows examples of peptides which may be used to generatemodified histone specific antibodies.

EXAMPLES Materials and Methods Collection and Preparation of BloodSamples

20 ml of blood were withdrawn by venepuncture into vacutainer tubescontaining sodium citrate, which were then kept on ice. Platelet richplasma (PRP) was prepared within 4 hours of blood collection bycentrifugation at 4° C. at 300 g for 20 minutes. An appropriate volumeof 20× inhibitor cocktail was added directly to the resultant PRP(resulting in supramaximal concentrations of okadaic acid, cypermethrin,staurosporine, trichostatin A, AEBSF, aprotinin, E-64, EDTA andleupeptin). Platelet poor plasma (PPP) was generated by centrifugationof the PRP on a Percoll underlay at 1500 g for a further 20 minutes.

Concentration of Plasma Samples

In some experiments, the nucleosomes in patient and normal plasmasamples were concentrated prior to analysis as follows:

Plasma samples (1.1 ml), collected and prepared as described above, werediluted with 2.4 ml of Dulbeccos PBS (not containing Ca²⁺ or Mg²⁺). Thesamples were centrifuged at ca. 328,000 g at 4° C. for 1.5 hours. Thesupernatants were removed and the pellets resuspended in 100 μl of 10 mMEDTA, vortexed and left at room temperature for 20 minutes. Cell lysisbuffer (190 μl), supplemented with the same inhibitor cocktail and 334μg/ml HBR-1 (heterophilic blocking reagent, Scantibodies LaboratoriesInc., San Diego), was added and incubated at room temperature for 1 hourprior to analysis by ELISA.

Note: the same ratio of EDTA:lysis buffer, containing the sameconcentrations of inhibitors and HBR-1, is used as the ELISA diluent forboth the patient samples and standard curve samples.

In some experiments, the standard curve was prepared in the presence ofa preparation of concentrated, pooled normal plasma.

ELISA on Nucleosomes From Human Blood Samples Method 1

A Nunc Maxisorp 96-well ELISA plate was coated overnight at 4° C. with apurified mouse anti-nucleosome monoclonal antibody at a concentration of2.5 μg/ml in a carbonate/bicarbonate buffer pH 9.5, 50 μl/well added(125 ng/well). The contents of the plate were flicked out and washedthree times with PBS (Dulbecco A). Blocking buffer (1% BSA in PBS+0.05%Tween 20) was then added.

PPP derived from normal blood which had been spiked with chickennucleosomes or buffer spiked with chicken nucleosomes, were dilutedappropriately with blocking buffer.

The block buffer was removed from the ELISA plate and diluted samples(for example 50 μl) were transferred to the plate. Appropriate controlwells were prepared.

The plate was sealed transferred to a shaking incubator (30° C.) for aperiod of 2 hours. The plate was flicked out and washed 4 times withPBS.

Anti-mark detection antibodies were appropriately diluted in blockbuffer and added to designated wells of the plate (typically 50μl/well). The plates were sealed and returned to the shaking incubatorfor a further 1.5 hours. The plates were washed as for the previousstep, followed by the addition of, for example, 50 μl of the anti-rabbitHRP conjugate to all wells, then returned to the incubator for 1 hour.

The wash step was repeated on the plates and 100 μl/well of SureBlue TMBMicrowell peroxidase substrate was added to all wells. The plates werereturned to the shaker/incubator to allow development of the bluecolour, typically for 40 minutes. Finally, the reaction was stopped bythe addition of TMB Stop Solution. The plates were read at a wavelengthof 450 nM.

Method 2

A modified indirect sandwich ELISA was used to detect covalentmodifications of histones of nucleosomes from human blood samples,concentrated as above.

A Nunc Maxisorp 96-well ELISA plate was coated overnight at 4° C. with apurified mouse anti-nucleosome monoclonal antibody at a concentration of3.0 μg/ml in a carbonate/bicarbonate buffer pH 9.5, 50 μl/well added(125 ng/well). The contents of the plate were flicked out and washedthree times with PBS (Dulbecco A). Blocking buffer (1% BSA inUltrablock+0.05% Tween 20) was then added for 1 hour.

Concentrated PPP samples were serially diluted with ELISA diluent.

The block buffer was removed from the ELISA plate and diluted samples(for example 50μl) were transferred to the plate. Appropriate controlwells were prepared.

The plate was sealed transferred to a shaking incubator (30° C.) for aperiod of 2 hours. The plate was flicked out and washed 4 times withPBS.

Detection antibodies were appropriately diluted and added to designatedwells of the plate (typically 50μl/well). The plates were sealed andreturned to the shaking incubator for a further 1.5 hours. The plateswere washed as for the previous step, followed by the addition of, forexample, 50 μl of biotinylated anti-rabbit conjugate to all wells, thenreturned to the incubator for 1 hour. The wash step was repeated andstreptavidin-HRP conjugate was added to all wells and the incubationcontinued for 0.5 hours.

The wash step was repeated on the plates and 100 μl/well of SureBlue TMBMicrowell peroxidase substrate was added to all wells. The plates werereturned to the shaker/incubator to allow development of the bluecolour, typically for 20 minutes. Finally, the reaction was stopped bythe addition of TMB Stop Solution. The plates were read at a wavelengthof 450 nM.

Results Spiking of Normal Blood With Nucleosomes

Using the sandwich ELISA described in Method 1 and an antibody todimethylated lysine 4 of histone H3 as the second antibody, samples ofbuffer spiked with chicken nucleosomes and PPP derived from normal bloodwhich had been spiked with chicken nucleosomes generated equivalentsignals (FIG. 1). No significant signal was obtained from PPP derivedfrom normal blood that had not been spiked with chicken nucleosomes overthe dilution range used in the assay (FIG. 1).

Spiking of Buffer With Nucleosomes

Using the sandwich ELISA described in Method 2 and an antibody todimethylated lysine 4 of histone H3 as the second antibody, samples ofbuffer spiked with chicken nucleosomes were shown to generate a standardcurve (FIG. 2).

Analysis of Patient Samples

Using the concentration method described above, the sandwich ELISAdescribed in Method 2 and an antibody to histone H3 dimethyl lysine 4 asthe second antibody in the ELISA, an increasing signal is measured asthe concentration of the sample derived from a patient increases (FIG.3). In contrast, a concentrated plasma sample from pooled normalindividuals, failed to generate a signal at the highest concentration(FIG. 3).

TABLE 1 Histone Residue Modification H3 Arg 2 Me H3 Arg 17 Me H3 Arg 26Me H3 Lys 4 Me H3 Lys 9 Me H3 Lys 14 Me H3 Lys 23 Me H3 Lys 27 Me H3 Lys36 Me H3 Lys 79 Me H3 Lys 9 Ac H3 Lys 14 Ac H3 Lys 18 Ac H3 Lys 23 Ac H3Lys 27 Ac H3 Lys 115 Ac H3 Lys 122 Ac H3 Ser 10 Phos H3 Ser 28 Phos H3Thr 3 Phos H3 Thr 11 Phos H3 Thr 118 Phos H4 Arg 3 Me H4 Arg 92 Me H4Lys 12 Me H4 Lys 20 Me H4 Lys 59 Me H4 Lys 79 Me H4 Lys 5 Ac H4 Lys 8 AcH4 Lys 12 Ac H4 Lys 16 Ac H4 Lys 20 Ac H4 Lys 77 Ac H4 Lys 79 Ac H4 Ser1 Phos H4 Ser 47 Phos H2A Lys 99 Me H2A Lys 5 Ac H2A Lys 9 Ac H2A Lys 13Ac H2A Lys 15 Ac H2A Lys 36 Ac H2A Lys 119 Ac H2A Ser 1 Phos H2A Lys 119Ubiq H2B Arg 99 Me H2B Lys 5 Me H2B Lys 23 Me H2B Lys 43 Me H2B Lys 5 AcH2B Lys 12 Ac H2B Lys 15 Ac H2B Lys 20 Ac H2B Lys 24 Ac H2B Lys 85 AcH2B Lys 108 Ac H2B Lys 116 Ac H2B Lys 120 Ac H2B Ser 14 Phos H2B Ser 32Phos H2B Ser 36 Phos H2B Lys 120 Ubiq H2A.X Ser 1 Phos H2A.X Ser 139Phos H2A.X Thr 136 Phos H2A.X Lys 119 Ubiq H2A.X Lys 5 Ac H2A.X Lys 9 AcH3.3 Arg 2 Me H3.3 Arg 17 Me H3.3 Arg 26 Me H3.3 Lys 4 Me H3.3 Lys 9 MeH3.3 Lys 14 Me H3.3 Lys 18 Me H3.3 Lys 27 Me H3.3 Lys 36 Me H3.3 Lys 37Me H3.3 Lys 79 Me H3.3 Lys 9 Ac H3.3 Lys 14 Ac H3.3 Lys 18 Ac H3.3 Lys23 Ac H3.3 Lys 27 Ac H3.3 Ser 10 Phos H3.3 Ser 28 Phos H3.3 Thr 11 Phos

TABLE 2 Histone Residue Modification H3 Arg 2 Me H3 Arg 17 Me H3 Arg 26Me H3 Lys 14 Me H3 Lys 23 Me H3 Lys 79 Me H3 Lys 9 Ac H3 Lys 14 Ac H3Lys 18 Ac H3 Lys 23 Ac H3 Lys 27 Ac H3 Lys 115 Ac H3 Lys 122 Ac H3 Ser10 Phos H3 Ser 28 Phos H3 Thr 3 Phos H3 Thr 11 Phos H3 Thr 118 Phos H4Arg 92 Me H4 Lys 12 Me H4 Lys 59 Me H4 Lys 79 Me H4 Lys 8 Ac H4 Lys 12Ac H4 Lys 16 Ac H4 Lys 20 Ac H4 Lys 77 Ac H4 Lys 79 Ac H4 Ser 1 Phos H4Ser 47 Phos H2A Lys 99 Me H2A Lys 5 Ac H2A Lys 9 Ac H2A Lys 13 Ac H2ALys 15 Ac H2A Lys 36 Ac H2A Lys 119 Ac H2A Ser 1 Phos H2A Lys 119 UbiqH2B Arg 99 Me H2B Lys 5 Me H2B Lys 23 Me H2B Lys 43 Me H2B Lys 5 Ac H2BLys 12 Ac H2B Lys 15 Ac H2B Lys 20 Ac H2B Lys 24 Ac H2B Lys 85 Ac H2BLys 108 Ac H2B Lys 116 Ac H2B Lys 120 Ac H2B Ser 32 Phos H2B Ser 36 PhosH2B Lys 120 Ubiq H2A.X Ser 1 Phos H2A.X Ser 139 Phos H2A.X Thr 136 PhosH2A.X Lys 119 Ubiq H2A.X Lys 5 Ac H2A.X Lys 9 Ac H3.3 Arg 2 Me H3.3 Arg17 Me H3.3 Arg 26 Me H3.3 Lys 4 Me H3.3 Lys 9 Me H3.3 Lys 14 Me H3.3 Lys18 Me H3.3 Lys 27 Me H3.3 Lys 36 Me H3.3 Lys 37 Me H3.3 Lys 79 Me H3.3Lys 9 Ac H3.3 Lys 14 Ac H3.3 Lys 18 Ac H3.3 Lys 23 Ac H3.3 Lys 27 AcH3.3 Ser 10 Phos H3.3 Ser 28 Phos H3.3 Thr 11 Phos

TABLE 3 H3 lys 4 (Me): ARTK(M)QTAR (SEQ ID NO: 1) H4 arg 3 (Me):SGR(M)GK (SEQ ID NO: 2) H4 lys 5 (Ac): SGRGK(A) (SEQ ID NO: 3)H3 lys 9 (Me): QTARK(M)STGV (SEQ ID NO: 6) H2B ser 14 (Phos):SAPAPKKGS(P)KK (SEQ ID NO: 7) H3 lys 27 (Me): AARK(M)SAPVCG(SEQ ID NO: 8) H3 lys 36 (Me): SGGVK(M)KPHKCG (SEQ ID NO: 9)H4 lys 20 (Me): RHRK(M)ILRDCG (SEQ ID NO: 10) H4 arg 3(Me)/lys 5(Ac):SGR(M)GK(A) (SEQ ID NO: 4) H4 Ser 2(phos)/Arg 3 (me)/Lys 5(Ac):S(P)GR(M)GK(A) (SEQ ID NO: 5)

REPRESENTATIVE HISTONE SEQUENCES Human H3 histone (SEQ ID NO: 11)ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA Human H4 histone (SEQ ID NO: 12)SGRGKGGKGLGKGGAKRHRKVLRDDIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKV FLENVIRDAVTYTEHAKRKTVTAMDVVYALKRQGRTLYG FGGHuman H2A histone (SEQ ID NO: 13)SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVY LAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPN IQAVLLPKKTESHHKAKGKHuman H2B histone (SEQ ID NO: 14)PEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK Human H2A.X histone (SEQ ID NO: 15)SGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGHYAERVGAGAPVY LAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGGVTIAQGGVLPNIQAVLLPKKTSATVGPKAPSGGKKATQASQEY Human H3.3 histone (SEQ ID NO: 16)ARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSAAIGALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA

1-22. (canceled)
 23. The method of assessing a cell-free nucleosomecomprising a histone modification in a sample, the method comprising:contacting a biological fluid sample with a first antibody that binds tocell-free nucleosomes, and; contacting cell-free nucleosomes bound tosaid first antibody with a second antibody which binds to a histonemodification.
 24. The method according to claim 23 wherein the firstantibody binds to a modified histone in said cell-free nucleosomes. 25.The method according to claim 23 wherein the histone modification isselected from the group consisting of acetylation, methylation,phosphorylation, ribosylation, citrullination, ubiquitination,hydroxylation, glycosylation, nitrosylation, glutamination andisomerisation.
 26. The method according to claim 23 wherein thebiological fluid sample is selected from the group consisting of blood,serum, plasma, lymph, blood fractions, urine, synovial fluid, spinalfluid and mucous.
 27. The method according to claim 23 wherein saidfirst antibody or said second antibody is immobilised.
 28. The methodaccording to claim 23 wherein the non-immobilised antibody of said firstantibody and second antibody comprises a detectable label.
 29. A methodof assessing a cell-free nucleosome comprising a histone modification ina sample, the method comprising: contacting a biological fluid samplewith a first antibody that binds to a histone modification, and;contacting cell-free nucleosomes bound to said first antibody with asecond antibody which binds to cell-free nucleosomes.
 30. The methodaccording to claim 29 wherein the second antibody binds to a modifiedhistone in said cell-free nucleosomes.
 31. The method according to claim29 wherein the histone modification is selected from the groupconsisting of acetylation, methylation, phosphorylation, ribosylation,citrullination, ubiquitination, hydroxylation, glycosylation,nitrosylation, glutamination and isomerisation.
 32. The method accordingto claim 29 wherein the biological fluid sample is selected from thegroup consisting of blood, serum, plasma, lymph, blood fractions, urine,synovial fluid, spinal fluid and mucous.
 33. The method according toclaim 23 wherein said first antibody or said second antibody isimmobilised.
 34. The method according to claim 23 wherein thenon-immobilised antibody of said first antibody and second antibodycomprises a detectable label.
 35. A method of assessing a diseasecondition in an individual, the method comprising: contacting abiological fluid sample obtained from the individual with a firstantibody that binds to cell-free nucleosomes, and; contacting cell-freenucleosomes bound to said first antibody with a second antibody whichbinds to a histone modification, wherein binding of said second antibodyto cell-free nucleosomes bound to said first antibody is indicative thatsaid individual has a disease condition.
 36. The method according toclaim 35 wherein the first antibody binds to a modified histone in saidcell-free nucleosomes.
 37. The method according to claim 35 wherein thehistone modification is selected from the group consisting ofacetylation, methylation, phosphorylation, ribosylation, citrullination,ubiquitination, hydroxylation, glycosylation, nitrosylation,glutamination and isomerisation.
 38. The method according to claim 35wherein the biological fluid sample is selected from the groupconsisting of blood, serum, plasma, lymph, blood fractions, urine,synovial fluid, spinal fluid and mucous.
 39. The method according toclaim 35 comprising determining a parameter selected from; the presenceof one or more cell-free nucleosomes comprising histone modifications inthe sample; an increase in the number of cell-free nucleosomescomprising histone modifications relative to normal levels; analteration in the ratio of one or more histone modifications incell-free nucleosomes in the sample relative to another histonemodification; and the presence of a threshold number of cell-freenucleosomes in the sample which comprise a histone modification, whereinsaid parameter is indicative that said individual has a diseasecondition.
 40. The method according to claim 35 wherein said firstantibody or said second antibody is immobilised.
 41. The methodaccording to claim 35 wherein the non-immobilised antibody of said firstantibody and second antibody comprises a detectable label.
 42. A methodof assessing a disease condition in an individual, the methodcomprising: contacting a biological fluid sample obtained from theindividual with a first antibody which binds to a histone modification,and; contacting cell-free nucleosomes bound to said first antibody witha second antibody that binds to cell free nucleosomes, wherein bindingof said second antibody to a nucleosome in said sample is indicativethat said individual has a disease condition.
 43. The method accordingto claim 42 wherein the second antibody binds to a modified histone insaid cell-free nucleosomes.
 44. The method according to claim 42 whereinthe histone modification is selected from the group consisting ofacetylation, methylation, phosphorylation, ribosylation, citrullination,ubiquitination, hydroxylation, glycosylation, nitrosylation,glutamination and isomerisation.
 45. The method according to claim 42wherein the biological fluid sample is selected from the groupconsisting of blood, serum, plasma, lymph, blood fractions, urine,synovial fluid, spinal fluid and mucous.
 46. The method according toclaim 42 comprising determining a parameter selected from; the presenceof one or more cell-free nucleosomes comprising histone modifications inthe sample; an increase in the number of cell-free nucleosomescomprising histone modifications relative to normal levels; analteration in the ratio of one or more histone modifications incell-free nucleosomes in the sample relative to another histonemodification; and the presence of a threshold number of cell-freenucleosomes in the sample which comprise a histone modification, whereinsaid parameter is indicative that said individual has a diseasecondition.
 47. The method according to claim 42 wherein said firstantibody or said second antibody is immobilised.
 48. The methodaccording to claim 42 wherein the non-immobilised antibody of said firstantibody and second antibody comprises a detectable label.